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PDBsum entry 2d4h
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Signaling protein
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PDB id
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2d4h
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References listed in PDB file
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Key reference
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Title
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How guanylate-Binding proteins achieve assembly-Stimulated processive cleavage of gtp to gmp.
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Authors
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A.Ghosh,
G.J.Praefcke,
L.Renault,
A.Wittinghofer,
C.Herrmann.
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Ref.
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Nature, 2006,
440,
101-104.
[DOI no: ]
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PubMed id
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Abstract
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Interferons are immunomodulatory cytokines that mediate anti-pathogenic and
anti-proliferative effects in cells. Interferon-gamma-inducible human guanylate
binding protein 1 (hGBP1) belongs to the family of dynamin-related large
GTP-binding proteins, which share biochemical properties not found in other
families of GTP-binding proteins such as nucleotide-dependent oligomerization
and fast cooperative GTPase activity. hGBP1 has an additional property by which
it hydrolyses GTP to GMP in two consecutive cleavage reactions. Here we show
that the isolated amino-terminal G domain of hGBP1 retains the main enzymatic
properties of the full-length protein and can cleave GDP directly. Crystal
structures of the N-terminal G domain trapped at successive steps along the
reaction pathway and biochemical data reveal the molecular basis for
nucleotide-dependent homodimerization and cleavage of GTP. Similar to effector
binding in other GTP-binding proteins, homodimerization is regulated by
structural changes in the switch regions. Homodimerization generates a
conformation in which an arginine finger and a serine are oriented for efficient
catalysis. Positioning of the substrate for the second hydrolysis step is
achieved by a change in nucleotide conformation at the ribose that keeps the
guanine base interactions intact and positions the beta-phosphates in the
gamma-phosphate-binding site.
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Figure 2.
Figure 2: Structural analysis of the GTPase reaction. a,
Comparison of the GppNHp  circle
Mg^2+- binding pockets of hGBP1^LG (blue) and hGBP1^FL (yellow)
highlights the dimerization-induced reorientation of the
catalytic Arg 48 and Ser 73 side chains on their corresponding
loops. The grey van der Waals surface of monomer B (black)
from the hGBP1^LG circle
GppNHp dimer is shown to indicate how Arg 48 of monomer A would
clash with Thr 133 from monomer B. b, GDP circle
AlF[3] from the Ras circle
RasGAP complex^16 (orange) is superimposed on GDP circle
AlF[3] from hGBP1^LG (green), indicating that the cis 'arginine
finger' of hGBP1 (R48) has a similar orientation to that of the
trans arginine from RasGAP (R789).
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Figure 4.
Figure 4: Structural analysis of the GDPase reaction. a,
Superimposition of the active sites of hGBP1^LG circle
GMP circle
AlF[4]^- (blue) and hGBP1^LG circle
GDP circle
AlF[3] (green) structures, respectively, showing the shift of
GMP for the second hydrolysis step. Black broken lines show
stabilizing polar interactions and red broken lines indicate
unfavourable vicinities between the nucleotide in the GDP circle
AlF[3] structure and the guanine cap residues as found in the
GMP
circle AlF[4]^- structure. b, Superimposition of
nucleotide-binding sites of hGBP1^LG circle
GMP (yellow, gold) and Ras circle
GDP (green; Protein Data Bank accession code 4Q21) structures
with the  -phosphate
of GMP occupying a similar position to that of the  -phosphate
of Ras circle
GDP. Arg 48 is pointing away from the active site. Red star
indicates possible steric hindrance between Lys 117 of the
(N/T)KxD motif from Ras and the GMP base conformation found in
hGBP1^LG.
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The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
Nature
(2006,
440,
101-104)
copyright 2006.
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Secondary reference #1
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Title
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Triphosphate structure of guanylate-Binding protein 1 and implications for nucleotide binding and gtpase mechanism.
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Authors
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B.Prakash,
L.Renault,
G.J.Praefcke,
C.Herrmann,
A.Wittinghofer.
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Ref.
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EMBO J, 2000,
19,
4555-4564.
[DOI no: ]
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PubMed id
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Figure 3.
Figure 3 Crystal contacts of hGBP1  GppNHp.
(A) The head-to-tail dimer A, which buries 2890 Å^2 of
surface area, where a lip from the LG domain is close to the
helical domain in helices 10
and the long helix 12.
The nucleotide is shown in ball and stick representation with
the yellow sphere representing the Mg^2+ ion. (B) The
head-to-head dimer B with 2140 Å^2 of buried surface,
using a similar colour code.
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Figure 4.
Figure 4 The phosphate-binding region and implications for GTP
hydrolysis. (A) Interactions of the phosphate oxygens and Mg^2+
with the P-loop (green), the switch I/phosphate cap (brown) and
the switch II region (maroon). Wat8 is in a homologous position
to the nucleophilic water found in other structures of
GTP-binding proteins. In contrast to those, there are three main
chain NH interactions of the protein with the  -phosphate.
(B) Potential catalytic residues around the active site that
could modify the rate of the GTPase reaction in an
oligomerization-dependent manner, without directly participating
in catalysis. (C and D) van der Waals surface representation of
the region of the active site of Ras (C) and hGBP1 (D) in the
GppNHp-bound state, the surface being coloured according to the
electrostatic potential, as calculated with GRASP (Nicholls et
al., 1991). In hGBP1, only the base is open to the solvent.
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The above figures are
reproduced from the cited reference
which is an Open Access publication published by Macmillan Publishers Ltd
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Secondary reference #2
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Title
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Structure of human guanylate-Binding protein 1 representing a unique class of gtp-Binding proteins.
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Authors
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B.Prakash,
G.J.Praefcke,
L.Renault,
A.Wittinghofer,
C.Herrmann.
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Ref.
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Nature, 2000,
403,
567-571.
[DOI no: ]
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PubMed id
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Figure 2.
Figure 2: Comparison of hGBP1 and Ras structures. a,
Superimposition of the LG domain of hGBP1 with the G domain of
Ras in complex with GDP(PDB accession no. 1Q21) as a stereo
view. N-terminal residues 1-36 of hGBP1 up to 1
have been omitted for clarity. The colour code is as in Fig. 1;
Ras is in cyan. b, Putative location of nucleotide-binding site
in hGBP1. The regions of hGBP1 potentially involved in binding
the guanine nucleotide are shown as obtained from a structural
superimposition of RasGDP (in cyan) with the corresponding
regions in hGBP1 (purple), highlighting functionally important
residues necessary for binding and conformational change as
balls or in ball-and-stick. Whereas Gly 60^ras overlays very
well with Gly 100^hGBP1, residues D119/D184 and T35/T75 do not.
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Figure 3.
Figure 3: Interaction of the C-terminal helix motif alpha- 12/13
with the helical and the LG domains. The electrostatic
surface potential shows that the highly charged regions of the
helical and LG domains are masked by an 12/13
motif, as indicated in the lower panel by showing 12/
13
in worm representation.
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The above figures are
reproduced from the cited reference
with permission from Macmillan Publishers Ltd
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